1. Field of the Invention
This invention relates to recombinant DNA technology for expressing heterologous proteins in bacteria. More particularly it relates to methods and means for efficient direct expression of prochymosin and mammalian growth hormones in Escherichia coli.
2. Description of the Prior Art
Calf rennin (chymosin) the preferred milk-clotting protease for use in cheese production is in short supply. Alternative milk-clotting agents; namely, fungal proteolytic enzymes have been developed. However, because of their great proteolytic activity, they tend to reduce the yield of cheese and often give bitter flavors.
The economic attractiveness of a steady and sufficient supply of a milk-clotting protease has led several investigators to apply recombinant DNA technology to the problem. Nishimoni et al., J. Biochem. 90:901-904 (1981) report cloning of the structural gene of prorennin (prochymosin) in E. coli. In a subsequent publication, Nishimoni et al., J. Biochem. 91:1085-1088 (1982), reported the nucleotide sequence of calf prorennin cDNA which they had cloned in E. coli. Construction of an expression plasmid having the lacUV5 promoter and its ability to produce a fused protein containing almost all of the prochymosin peptide joined to the short N-terminal peptide of E. coli beta-galactosidase is described by Nishimoni et al., Gene 19:337-344 (1982).
European Patent Application No. 73,029 published Mar. 2, 1983 describes calf prorennin DNA-containing plasmids, microorganisms (E. coli) transformed with said plasmids, and their expression of prorennin.
British Patent Application No. 2,091,271A, published July 28, 1982, discloses methods and agents for producing rennin, prorennin and preprorennin, including the use of various promoters (lac, trp, ura3, etc.) to obtain expression. One of the disclosed DNA sequences that codes for preprorennin has attached to it a transcriptional promoter and a ribosomal binding site at the 5'-end. The distance between the beginning of the DNA which codes for preprorennin and the DNA segment carrying the transcriptional promoter and ribosomal binding site is varied.
British Patent Application No. 2,100,737A, published Jan. 6, 1983, describes recombinant DNA technology for producing chymosin, methionine chymosin, prochymosin, methionine prochymosin and preprochymosin. Vectors carrying an E. coli trp promoter-operator fragment and a transcription terminator, an initiation codon and a Shine-Dalgarno (SD) sequence which serves as a ribosome binding site are disclosed. Investigation of the effect of spacing between the SD and ATG sequences is also disclosed.
European Patent Application No. 77,109, published Apr. 20, 1983, describes DNA molecules, i.e., plasmids, comprising genes for preprochymosin and specific DNA sequences such as a double lac UV5 or a modified trp system, and their use to transform microorgamisms (lactobacilli, streptococci, bacillus or yeast) to generate transformants which produce preprochymosin in its allelic and maturation forms.
European Patent Application No. 36,776, published Sept. 30, 1981 describes expression vectors having the trp promoter-operator from which the attenuation region has been deleted, and methods for their production. Transformants carrying said vectors can be grown up in tryptophan-rich media so that cell growth proceeds uninhibited by premature expression of heterologous peptide encoded by an insert otherwise under control of the trp promoter-operator system.
Emtage et al., Proc. Natl. Acad. Sci. 80:3671-3675, 1983 and Japanese Patent Application No. SHO 58-38,439, filed Mar. 9, 1983, communicated to us by Beppu, describe construction of hybrid plasmids carrying prochymosin cDNA and containing the E. coli trp operon and the use thereof for expression of prorennin at levels greater than those reported by prior investigators. A further communication from Beppu disclosed an amendment to said Japanese application, said amendment being filed on Nov. 15, 1983. The amendment relates, in part, to the effect of variation in the distance separating the SD sequence from the initiation codon for prochymosin, and the effect of replacing the N-terminal amino acids of prochymosin by peptides of varying length.
Harris et al., Nucleic Acids Research 10:2177-2187 (1982) report the cloning and nucleotide sequence of cDNA coding for preprochymosin. Goff et al., Gene 27, 35-46 (1984) describe the expression of calf prochymosin in Saccharomyces cerevisiae, a yeast. The restriction endonuclease cleavage, map and DNA sequence of preprochymosin cDNA have been published [Nishimoni et al., J. Biochem. 91:1085-1088, (1982)].
Mammalian growth hormones, including human epidermal growth factor (h-EGF), are of considerable interest because of their potential to improve animal husbandry. Their general use has been restricted because of their very limited availability. The economic attractiveness of an adequate supply of said hormones has led several investigators to apply recombinant DNA technology to the problem.
Human epidermal growth factor (EGF) or urogastrone is not only a stimulator of epidermal tissue growth but is also a potent inhibitor of gastric acid secretion. The full potential of EGF has not been investigated primarily because of lack of sufficient material.
The use of recombinant DNA methodology for the manufacture, cloning and expression of a structural gene for urogastrone and of genes for polypeptide analogs thereof are described in International Patent Application No. 83/04030, published Nov. 24, 1983.
The cloning of DNA complementary to bovine growth hormone mRNA, the nucleotide sequence thereof and the corresponding amino acid sequence predicted therefrom are reported by Miller et al., in European Patent Application No. 47,600, published Mar. 17, 1983 and J. Biol. Chem. 255, 7521-7524 (1980), and by Woychik et al. in Nucleic Acids Research 10, 7197-7210 (1982). British Patent Application No. 2,073,245A, published Oct. 14, 1981, and Kesket et al., Nucleic Acids Research 9, 19-30 (1981) describe the cloning of bovine growth hormone and its expression in E. coli HB101 as a fused beta-lactamase-bovine growth hormone protein.
Processes for expressing bovine growth hormone gene, plasmids and plasmid hosts for use therein are disclosed in European Patent Application Nos. 67,026 and 68,646, published Dec. 15, 1982 and Jan. 5, 1983, respectively. Each application discloses E. coli as the host organism. The latter application, the counterpart of U.S. Pat. No. 4,443,539 issued Apr. 17, 1984, also divulges Saccharomyces cerevisiae as host organism.
Seeburg et al., DNA, 2 37-45 (1983) report the cloning in bacteria of cDNAs prepared using poly (A)mRNA from bovine or porcine pituitaries and the construction of expression vectors thereform which achieved efficient bacterial production of the mature animal (bovine or porcine) growth hormones. The technique adopted was analogous to that previously described by Goeddel et al., Nature, 281, 544-548 (1979) for direct expression of human growth hormone in E. coli. In each instance the bacterial expression vectors used were under control of the E. coli trp promoter. European Patent Application Nos. 103,395 and 104,920, published Mar. 21, 1984 and Apr. 4, 1984, describe production of bovine growth hormone-like polypeptide and production of swine growth hormone-like polypeptides, respectively via recombinant DNA methodology.
Administration of bovine growth hormone to dairy cows increases milk production and improves the feed intake to milk output ratio [Macklin, J. Dairy Science 56, 575-580 (1973)]. European Patent Application No. 85,036A, published Aug. 3, 1983, discloses that biosynthetically produced (by rDNA) bovine growth hormone and/or fragments of it also increase milk production in cows and production of meat, wool, eggs and fur in pigs and other farm animals.
U.K. Patent Specification No. 1,565,190, published Apr. 16, 1980, discloses recombinant plasmid vectors capable of transforming microorganims and containing within their nucleotide sequences subsequences which code for the growth hormone of an animal species. U.S. Pat. No. 4,237,224 describes plasmid vectors for introducing foreign DNA into unicellular organisms.
Plasmids having a HindIII insertion site for a chosen eukaryotic DNA fragment, said site being adjacent to a bacterial promoter such as the trp promoter, wherein the transcription and translation of the DNA fragment are controlled by the promoter, are described in U.S. Pat. No. 4,349,629.
The level of expression of a cloned gene is influenced by a number of factors such as the number of gene copies and the efficiency of transcription and translation. Efficient transcription of an inserted gene requires the presence of a strong promoter and efficient translation requires the presence of a suitable ribosome binding site in the mRNA and appropriate spacing between the rbs and the translation initiation codon. The promoter precedes that portion of the DNA (structural gene) which codes for a protein. The ribosome binding site (rbs), or ribosome recognition sequence, is believed to consist of a sequence at least 3-9 bp long, known as the Shine-Dalgarno (SD) sequence. It begins 3 to 11 bp upstream from the AUG which encodes the amino terminal methionine of the protein [Guarante et al., Cell 20:543-553, (1980)], and is complementary to the 3'-terminal sequence of 16S RNA.
The separation of the promoter from the translational start signal (AUG) for a gene can markedly affect the levels of protein produced (Guarante et al., loc. cit. and references cited therein). This reference and Ptashne et al., U.S. Pat. No. 4,332,892 issued June 1, 1982 describe the effects of placing a "portable promoter" fragment at varying distances from the 5'-end of a gene upon expression.
Other references relevant to the effect of defined alterations of nucleotide sequences and especially of variations between the SD region and the start codon are: Scherer et al., Nucl. Acids Res. 8:3895-3907 (1980); Shepard et al., DNA 1:125-131 (1982); Windass et al., Nucl. Acids Res. 10:6639-6657 (1982); De Boer et al., DNA 2:231-235 (1983); Tacon et al., Molec. gen. Genet. 177, 427-438 (1980); and Itoh et al., DNA 3, 157-165 (1984).